Fluorescence characteristics of peroxidation products in porcine intestinal brush-border membranes

Treatment of the porcine intestinal brush-border membranes with 100 microM ascorbic acid and 10 microM Fe2+ in the presence of various concentrations of tert-butyl hydroperoxide (t-BuOOH) resulted in a marked fluorescence development at 430 nm, depending on the hydroperoxide concentration. This fluorescence formation was closely related to lipid peroxidation of the membranes as assessed by formation of conjugated diene. However there is no linear relation between thiobarbituric acid-reactive substances (TBARS) and fluorescence formation. On the other hand, fluorescence formation in the membranes by treatment with ascorbic acid/Fe2+ or t-BuOOH alone was negligible. The results with antioxidants and radical scavengers suggest that ascorbic acid/Fe2+/t-BuOOH-induced lipid peroxidation of the membranes is mainly due to t-butoxyl and/or t-butyl peroxy radicals. Most TBARS produced during the peroxidation reaction were released from the membranes, but fluorescent products remained in the membrane components. The fluorescence properties of products formed by lipid peroxidation of the membranes were compared with those of products derived from the interaction of malondialdehyde (MDA) or acetaldehyde with the membranes. The fluorescence products in the acetaldehyde-modified membranes also exhibited the emission maximum at 430 nm, while the emission maximum of MDA-modified membranes was 470 nm. The fluorescence intensity of MDA-modified membranes was markedly decreased by treatment with 10 mM NaBH4 but that of the peroxidized or acetaldehyde-modified membranes was enhanced by about two-fold with the treatment. In addition, a pH dependence profile revealed that the fluorescence intensity of the peroxidized or acetaldehyde-modified membranes decreases with increasing pH of the medium, whereas that of MDA-modified ones did not change over the pH range from 5.4 to 8.0. On the basis of these results, the fluorescence properties of products formed in the intestinal brush-border membranes by lipid peroxidation are discussed.     

Spotted fever group rickettsia in dogs in Japan

Prevalence of antibody against spotted fever group-rickettsia in dogs (14/134) from the northern part of Shikoku Island, where spotted fever group rickettsia infection in human is endemic, is significantly higher than that in dogs (4/189) from nonendemic areas.     

Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin, and collagen and characterization of its three distinct functional sites

We have used purified proteolytic fragments of von Willebrand factor (vWF) to characterize three related functional sites of the molecule that support interaction with platelet glycoprotein Ib, collagen, and heparin. A fragment of 116 kDa was found to be dimeric and consisted of disulfide-linked subunits which, after reduction and alkylation, corresponded to the previously described 52/48-kDa fragment extending from residue 449 to 728. Fragment III-T2, also a dimer, was composed of two pairs of disulfide-linked subunits, two 35-kDa heavy chains (residues 273-511) and two 10-kDa light chains (residues 674-728). The 116-kDa fragment, but not the constituent 52/48-kDa subunit, supported ristocetin-induced platelet aggregation and retained 20% (on a molar basis) of the ristocetin cofactor activity of native vWF; fragment III-T2 retained less than 5% activity. All three fragments, however, inhibited vWF interaction with glycoprotein Ib. Both 116-kDa and 52/48-kDa fragments inhibited vWF binding to heparin with similar potency, while fragment III-T2 had no effect in this regard. Only the 116-kDa fragment inhibited vWF binding to collagen. These results indicate that dimeric fragments containing two glycoprotein Ib-binding sites possess the minimal valency sufficient to support ristocetin-induced aggregation. The sequence comprising residues 512-673, missing in fragment III-T2, is necessary for binding to heparin and collagen and may be crucial for anchoring vWF to the subendothelium. Immunochemical and functional data suggest that the same sequence, although not essential for interaction with glycoprotein Ib, may influence the activity of the glycoprotein Ib-binding site. Only binding to collagen has absolute requirement for intact disulfide bonds. Thus, the three functional sites contained in the 116-kDa domain of vWF are structurally distinct.     

Dynamic conformational changes of 21 S dynein ATPase coupled with ATP hydrolysis revealed by proteolytic digestion

Conformational changes of 21 S dynein ATPase from sea urchin sperm flagella were examined by tryptic digestion under physiological conditions. In the presence of 2 mM ATP or ADP plus 100 microM inorganic vanadate (Vi), dynein heavy chains were digested by trypsin into quite different polypeptides from those obtained in other cases (no addition, 2 mM ATP, 4 mM adenosine 5'-(beta,gamma-imido)triphosphate, 4 mM adenosine 5'-(beta,gamma-methylene)triphosphate, 2 mM ADP, 100 microM Vi). In the presence of 4 mM adenosine 5'-O-(3-thiotriphosphate), however, the digestion pattern was similar to that in the presence of ATP (ADP) and Vi, to a certain extent. In all conditions other than the presence of ATP (ADP) and Vi, 165- and 135-kDa polypeptides were the main products, whereas in the presence of ATP (ADP) and Vi, 200-, 150/148-, and 105/96-kDa peptides were produced and 320-kDa peptide became rather inaccessible to trypsin. The latter digestion pattern was not observed in the absence of divalent cations. These results suggest that, in the ATP hydrolysis cycle, dynein changes its conformation remarkably in the dynein-ADP-Pi state, which is presumably responsible for force generation.     

Characteristics of motor apparatus in testicular, epididymal and ejaculated spermatozoa. A study using demembranated sperm models

Morphological alterations of cytoplasmic organelles occur in the early stage of the cell fusion reaction induced by HVJ (Sendai virus) and these alterations are reversed in the late stage of the reaction. When the cells were treated with the virus at 37 °C for 3 min, the mitochondria became condensed, the cisternae of the endoplasmic reticula and Golgi stacks became highly distended, the inner space of the nuclear envelopes became enlarged, and the arrangement of 100 Å filaments became disordered. All these morphological changes were gradually reversed during further incubation at 37 °C and had been completely reversed by the end of the cell fusion reaction. These changes occurred at the time of interaction of the cell membrane with HVJ, and seemed to represent functional responses of the living cell to perturbation of the membrane resulting from its interaction with the virus.     

The effects of 5% carbon dioxide on the quantitative analysis of long-term pathology of the brain after surface hypothermia

To evaluate the effects of 5% carbon dioxide (CO2) administration for hypothermic circulatory arrest, neurological evaluation and pathological studies were carried out on the canine brain. Twenty-two dogs were assigned to five groups: Group 1: Three dogs without hypothermia were sacrificed as the control group. Group 2: Nine dogs were subjected to surface hypothermia (20 degrees C) under deep ether anesthesia with 100% oxygen (O2) and hyperventilation. Circulatory arrest time was 30 min in Group 2A and 60 min in Group 2B. Group 3: Ten dogs were surface cooled (20 degrees C) under deep ether anesthesia with a 95% O2 and 5% CO2 mixture. Thirty minutes of circulatory arrest was instituted in Group 3A and 60 min in Group 3B. Dogs in Groups 2 and 3 were surface rewarmed and kept alive until they were sacrificed electively 6 or more months later. Results were as follows: (i) Postoperative neurological disturbance was detected in only two dogs in Group 2B. (ii) The percentage of damaged nerve cells among the total nerve cells counted in the cerebral cortex of the frontal lobe was significantly greater in Groups 2A (22.4%), 2B (30.1%), 3A (19.6%), and 3B (22.2%) compared with Group 1 (7.1%). (iii) The number of glia cells per nerve cell in the cerebellar dentate nucleus was significantly higher in Group 2B (27.2) than in Groups 1 (11.8), 2A (16.7), 3A (17.9), and 3B (18.6). (iv) The number of Purkinje cells in a 10-mm length of the cerebellum was markedly reduced to 89 in Group 2B compared with 122, 134, and 117 in Groups 1, 2A, and 3A, respectively. In conclusion, the results of quantitative pathological brain analysis reflected the incidence of postoperative neurological disturbance and suggested that the administration of 5% CO2 could prolong the time limit for circulatory arrest.     

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella coupled with ATP hydrolysis

Conformational changes of the beta chain of the outer-arm dynein from sea urchin sperm flagella in relation to ATP hydrolysis was examined by tryptic digestion. Tryptic digestion of the beta chain in the presence of 2 mM ATP (ADP) and 100 microM vanadate (Vi) or in the presence of 4 mM ATP gamma S produced different polypeptides from in the case of no addition. The difference was similar to the result previously reported for 21S outer-arm dynein heavy chains [Inaba, K. & Mohri, H. (1989) J. Biol. Chem. 264, 8384-8388]. Unlike the tryptic digestion pattern of 21S dynein heavy chains, however, the 135-kDa polypeptide was consistently produced from the beta chain, even in the presence of ATP (ADP) and Vi. The tryptic digestion pattern of the 21S particle reconstituted from the separated a chain, the beta/IC1 complex and the IC2/IC3 complex [Tang, W.-J.Y., Bell, C.W., Sale, W.S., & Gibbons, I.R. (1982) J. Biol. Chem. 257, 508-515] was similar to that of intact 21S dynein; the 135-kDa polypeptide was only slightly produced in the presence of ATP and Vi. The digestion rate constant of the 135-kDa polypeptide from the beta chain in the presence of ATP and Vi was significantly decreased as compared with in the case of 21S dynein or that of the reconstituted 21S particle. These results suggest that the trypsin sensitivity of the 135-kDa region of the beta chain changes with the association of the beta/ICI complex with the alpha chain and the IC2/IC3 complex in the presence of ATP and Vi.     

Different conformation changes induced by calcium and terbium of the porcine intestinal calcium-binding protein

The fluorescence of 1-anilino-8-naphthalenesulfonate (ANS) is progressively enhanced with increasing concentration of it, showing a proportionate blue shift of the emission maximum, by the interaction with the porcine intestinal Ca2+-binding protein (CaBP) in the absence of Ca2+. The apo-CaBP has a single binding site for ANS as determined by the fluorescence change, the apparent dissociation constant (Kd) estimated at 49.1 microM. Addition of Ca2+ or Tb3+ to the ANS-apo-CaBP system is capable of enhancing its fluorescence up to about 2- or 5-fold, respectively, causing further blue shift of the emission maximum. These metal ions do not affect the capacity of ANS binding, but Ca2+ slightly increases the Kd value. Increase of the fluorescence of the ANS-CaBP complex by increasing binding of Ca2+ to it was monophasic, while that with Tb3+ was biphasic, both saturated at the same molar ratio, 2, of added cations to the complex. Biphasic change of response has also been observed in UV absorption of the CaBP with increasing concentration of Tb3+. With a half-saturating concentration of Tb3+, Ca2+ can induce a much higher enhancement of the ANS fluorescence than excess Ca2+ alone. All these results indicate that the CaBP molecule contains a single ANS binding site and the conformation and/or microenvironment surrounding bound ANS of the protein is altered reversibly with binding of Ca2+ or Tb3+ to it and that there are differences between Ca2+- and Tb3+-induced conformation changes around the ANS-binding site and the tyrosine residue of it.     

Effects of hydrolase inhibitors on fertilization of sea urchins: I. Protease inhibitors

To search the spermatozoa of sea urchins for their lysins, the eggs were inseminated in the presence of various protease inhibitors. Among them, two chymotrypsin-specific inhibitors, chymostatin and N-tosyl-L-phenylalanyl-chloro-methane, as well as p-nitrophenyl p′-guanidinobenzoate, inhibit fertilization of the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus intermedius. A chymotrypsin-like protease is presumed to be a lysin of the sea urchins, since the inhibition of fertilization by chymostatin is remarkably diminished if the eggs are pretreated with trypsin or chymotrypsin to break the vitelline coat before insemination, and since N-tosyl-L-phenylalanyl-chloromethane, and p-nitrophenyl p′-guanidinobenzoate, as well as chymostatin, inhibit the fertilization. In all the sea urchins so far studied, elevation of fertilization envelopes is inhibited by leupeptin, antipain, soybean trypsin inhibitor, and p-nitrophenyl p′-guanidinobenzoate, all of which are potent trypsin inhibitors. Synthetic inhibitors have cytotoxic side effects on the eggs, but the microbial and plant inhibitors have no such effects.     

Estrogen and bisphenol A disrupt spontaneous [Ca(2+)](i) oscillations in mouse oocytes

The present work aims to study the effects of estrogen or endocrine disrupters (EDs) on the dynamic changes in intracellular Ca(2+) concentration of mouse immature oocytes (IOs) loaded with Ca(2+)-sensitive dye Fura-2 using an image analyzer. The majority of IOs isolated from the ovary exhibited spontaneous Ca(2+) oscillations at regular intervals. Entry of external Ca(2+), probably through gap junctions, contributes to Ca(2+) oscillations since they were reversibly inhibited by removing Ca(2+) from the bathing medium or by the application of a gap-junction inhibitor carbenoxolone (CBX, 30 microM). Both 17beta-estradiol (E2) and E2-BSA, a membrane impermeable estrogen, shortened the duration of Ca(2+) oscillations in a dose-dependent manner (1-1000 nM), and produced an irregular pattern of the oscillations, strongly suggesting that E2 acts on the plasma membrane of the oocyte. For bisphenol A (BPA), one of the estrogen-mimicking EDs, a 10,000-fold higher concentration (100 microM) was necessary to exert similar inhibitory action to that of E2.